Mobile Protons Limit the Stability of Salt Bridges in the Gas Phase: Implications for the Structures of Electrosprayed Protein Ions.

Electrosprayed protein ions can retain native-like conformations. The intramolecular contacts that stabilize these compact gas-phase structures remain poorly understood. Recent work has uncovered abundant salt bridges in electrosprayed proteins. Salt bridges are zwitterionic BH/A contacts. The low dielectric constant in the vacuum strengthens electrostatic interactions, suggesting that salt bridges could be a key contributor to the retention of compact protein structures. A problem with this assertion is that H are mobile, such that H transfer can convert salt bridges into neutral B/HA contacts. This possible salt bridge annihilation puts into question the role of zwitterionic motifs in the gas phase, and it calls for a detailed analysis of BH/A versus B/HA interactions. Here, we investigate this issue using molecular dynamics (MD) simulations and electrospray experiments. MD data for short model peptides revealed that salt bridges with static H have dissociation energies around 700 kJ mol. The corresponding B/HA contacts are 1 order of magnitude weaker. When considering the effects of mobile H, BH/A bond energies were found to be between these two extremes, confirming that H migration can significantly weaken salt bridges. Next, we examined the protein ubiquitin under collision-induced unfolding (CIU) conditions. CIU simulations were conducted using three different MD models: (i) Positive-only runs with static H did not allow for salt bridge formation and produced highly expanded CIU structures. (ii) Zwitterionic runs with static H resulted in abundant salt bridges, culminating in much more compact CIU structures. (iii) Mobile H simulations allowed for the dynamic formation/annihilation of salt bridges, generating CIU structures intermediate between scenarios (i) and (ii). Our results uncover that mobile H limit the stabilizing effects of salt bridges in the gas phase. Failure to consider the effects of mobile H in MD simulations will result in unrealistic outcomes under CIU conditions.